Bile acid signaling plays a critical role in control of liver metabolism and inflammation. Accumulation of toxic bile acids causes liver inflammation and contributes to pathogenesis of chronic non-alcoholic fatty liver disease (NAFLD), diabetes and obesity. The rate of bile acid synthesis is regulated by the first and rate- limiting enzyme, cholesterol 7?-hydroxylase (CYP7A1), whereas bile acid composition is regulated by sterol 12?-hydroxylase (CYP8B1) in cholic acid synthesis. Bile acids activate a nuclear receptor FXR and a G protein coupled receptor TGR5 to regulate lipid, glucose and energy metabolism. It has been proposed that FXR plays a key role in bile acid feedback inhibition of bile acid synthesis. The anti-inflammatory action of bile acids has been recognized recently. However, the underlying molecular mechanisms of bile acid regulation of hepatic metabolic homeostasis and inflammation are not understood. Type 2 diabetes patients have higher serum 12?-hydroxylated bile acids, which is correlated to insulin resistance and dyslipidemia. Three specific aims are designed to test the hypothesis that FXR and TGR5 signaling regulate bile acid synthesis and composition, which affect hepatic inflammation, insulin resistance, NAFLD and obesity. Specific aim 1 is to study the mechanisms of FXR and TGR5 signaling in bile acid metabolism. FXR-/-, TGR5-/- and FXR/TGR5 double knockout (DK) mice will be used to test the hypothesis that the FXR/TGR5/CYP8B1 pathway may play a role in regulation of bile acid synthesis and lipid metabolism. Specific aim 2 is to study TGR5 and FXR signaling in anti-inflammation in hepatocytes. Activation of FXR and TGR5 signaling may affect macrophage polarization to reduce hepatic inflammation. TGR5-/-, FXR-/- and FXR/TGR5 DK mice will be used to study the mechanisms of anti-inflammatory action of FXR and TGR5. Specific aim 3 is to study nutrient regulation of the diurnal rhythm and fasting/restricted feeding on bile acid synthesis in NAFLD, insulin resistance and obesity. Fasting and restricted feeding and high fat diet affect the circadian rhythm of bile acid synthesis and energy metabolism. Experiments are designed to study the role of CYP8B1 in dyslipidemia, insulin resistance and NAFLD. This study is highly significant in elucidating the molecular mechanisms of regulation of bile acid synthesis and lipid homeostasis, and pathogenesis of liver-related metabolic diseases. Results from this study will have potential for developing drug therapies to improve insulin sensitivity, liver inflammation, hepatic steatosis, diabetes and obesity.